Aspects of endocrine disruption in the
Upper Olifants River and Lake Loskop
Christoff Truter
Stellenbosch University
Presentation to the Olifants River Forum
7 December 2012
The endocrine system
• Communication network
Hormones = Signals
–
–
–
–
–
Reproduction/ sexual characteristics
Growth and development
Metabolism
Immunity
Behaviour
The endocrine system: Basic mechanism of action
Hormones (testosterone)
fitnessanddefense.com
Testicle
Cell
Hormone receptor
Nucleus
DNA
Glands
Target tissues
Transcription + Translation
Bloodstream
Phenotypic change
Hormones in the aquatic environment
Natural and synthetic
•
•
•
•
Oral contraceptives
Hormone replacement therapy
Growth stimulators (Agriculture)
Human and animal excretes
unifiedlifestyle.com
Activity at parts per trillion concentration
 1 teaspoon in 50 million litres
sexmedsonline.com
toptenz.net
Impacts on fish
• Impaired reproduction
–
–
–
–
Intersex fish (Jobling et al. 1998)
Reduced egg production (Brion et al. 2001)
Skewed sex ratios (Nimrod and Benson 1998)
Altered oogenesis (Woodling et al. 2006)
Gakuranman.com
Whole lake experiment
Kid et al. 2007
Synthetic estrogen (Ethinylestradiol)
• 3 years exposure (5 ng/L)
– Fathead minnow population
• Male fish feminised
• Altered oogenesis in females
Fathead minnow
– Population collapse
denr.sd.gov
Ovary
Normal female
Exposed female
Testis
Normal male
Exposed male
Upper Olifants River
Oberholster et al. 2010
Upper Olifants River: Human impacts
Agriculture
Acid mine drainage
thesanitationcrisis.wordpress.co.za
Industrial effluents
Informal settlements
Sewage works effluents
Aims
• Determine female hormone concentrations
in the Upper Olifants River
– Assess seasonal and spatial variation
17-β-estradiol
Ethinylestradiol
Water collection
• 6 Localities (Sub-surface samples)
–
–
–
–
Autumn (2011)
Winter (2011)
Spring (2011)
Summer (2011)
Study sites
Oberholster et al. 2010
Water extraction and analysis
• C18 Solid Phase Extraction of organic compounds
• 17-β-Estradiol
• Ethinylestradiol
– Immunoassays (Commercial ELISA kits)
17-β-Estradiol
May 2011
June 2011
September 2011
November 2011
35
17-β-estradiol (ng/L)
30
25
20
15
10
5
0
Davel
Steenkool
Spruit
Olifants
River View
SID
Gorge
Mean ± Standard Deviation (technical repeats)
17-β-Estradiol
May 2011
June 2011
September 2011
November 2011
35
17-β-estradiol (ng/L)
30
US EPA Maximum Acceptable
Toxicant Concentration (MATC)
25
20
15
US EPA predicted no-effect concentration (Fish)
Effects:
• Egg production
• Male secondary characteristics
• Gonad histology
10
5
0
Davel
Steenkool
Spruit
Olifants
River View
SID
Gorge
Mean ± Standard Deviation (technical repeats)
Ethinylestradiol
Ethinylestradiol (ng/L)
May 2011
June 2011
September 2011
November 2011
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
Davel
Steenkool
Spruit
Olifants
River View
SID
Gorge
Mean ± Standard Deviation (technical repeats)
Ethinylestradiol
Ethinylestradiol (ng/L)
May 2011
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
June 2011
September 2011
Complete feminization of fathead minnows
(Lange et al. 2001)
November 2011
US EPA Maximum Acceptable
Toxicant Concentration (MATC)
Effects:
• Impaired fertilization success
US EPA predicted no-effect concentration (Fish)
Davel
Steenkool
Spruit
Olifants
River View
SID
Gorge
Mean ± Standard Deviation (technical repeats)
30
25
20
15
10
5
0
-5
a
ab
ab
ab
b
ab
Ethinylestradiol (ng/L)
17-β-estradiol (ng/L)
Spatial variation among localities
10
8
6
4
2
0
-2
Mean ± Standard Deviation
Pairwise differences assessed with Kruskal Wallis ANOVA
Seasonal variation
across the Upper Olifants System
7
Ethinylestradiol (ng/L)
17-β-estradiol (ng/L)
25
20
15
10
5
0
6
5
4
3
2
1
0
May
June
Sept
Nov
May
June
Sept
Nov
Mean ± Standard Deviation
Pairwise differences assessed with Kruskal Wallis ANOVA
Conclusions
• Potential disruption of the gonadal endocrine axis
throughout the system
• Potential reproductive impairment at five of the six sites
Future work
In vivo:
• Expose juvenile Mozambique tilapia to water collected
during summer
– Expression of a selection of genes associated with the reproductive
system
Lake Loskop Mozambique tilapia
• Metabolic disorders
– Obesity and lipid peroxidation (pansteatitis) (Dabrowski et al. unpublished)
Lake Loskop
Allan Stratton
Oberholster et al. 2011
Lake Loskop Mozambique tilapia
Metabolic disorders
Thyroid disruption
Reviewed in Casals-Casas and Desvergne 2011
•
Loskop Fish: Hiperthyroidism relative to Lake Flag Boshello (Dabrowski et al. unpublished)
– Potential links to thyroid disruption by endocrine disrupting chemicals
• Industrial chemicals?
• Metals?
• Cyanotoxins (microcystins)?
• Other?
Further investigation needed:
Pathway specific investigation of the Hypothalamus-pituitary-thyroid endocrinal axis
The Thyroid Cascade
Hypothalamus
Thyrotrophin releasing hormones (TRH)
Pituitary
Thyroid-stimulating hormones (TSH)
Thyroid gland
Thyroid hormones (T3 and T4)
Wikipedia.org
Target tissues
Thyroid Receptors (TR alpha and TR beta)
Transcription (Thyroid activity)
Growth
Development
Metabolic regulation
Aims
• Investigate thyroid disruption in fish caught
from Lake Loskop using gene expression
based biomarkers
– mRNA expression
• TR alpha
• TR beta
Fish collection (August 2012)
• Mozambique tilapia
– Lake Loskop (Gill nets)
– Stellenbosch University Aquaculture Department
RNA isolation
Brains tissue
• Lake Loskop
– 5 males
– 3 females
• Stellenbosch
– 3 males
– 3 females
Gene expression (RT-qPCR)
• Thyroid Receptor-α
• Thyroid Receptor-β
• Reference gene: β-actin
– Relative quantification: 2-(ΔΔCt)-method (Pfaffl 2001)
Mozambique tilapia
Lake Loskop vs Stellenbosch University
Relative expression of mRNA
2
1.8
1.6
1.4
1.2
1
0.8
Lake Loskop
**
Stellenbosch
0.6
0.4
0.2
0
8
6
TR alpha
8
6
N
TR beta
Mean ± Standard Deviation
* P = 0.02; student’s t-test
Gender effects on TR expression
TR alpha
1.4
1.2
1
0.8
Lake Loskop
0.6
Stellenbosch
0.4
0.2
0
5
3
Male
TR beta
2.5
3
3
Female
N
Relative expression of mRNA
Relative expression of mRNA
1.6
2
1.5
1
0.5
0
5
Male
3
3
3
Female
Mean ± Standard Deviation
Pairwise differences assessed with student’s t-test
N
Cyanobacteria (cyanotoxins) as culprit?
96 hour Zebrafish larvae exposure (Yan et al. 2012)
Microcystin-LR (μg/L)
Microcystin-LR (μg/L)
Lake Loskop: August 2012 (Oberholster and Cheng, unpublished data)
•
Water
– Riverine zone - 1.299 mg/L
– Transitional zone - 1.302 mg/L
– Lacustrine zone - 1.401 mg/L
•
Mozambique tilapia
– Livers: 0.075 μg/kg
– Muscle: below detection limit
Future work
• Measure TSHβ expression in adult Mozambique tilapia brains
Yan et al. 2012
Microcystin-LR (μg/L)
• Expose juvenile Mozambique tilapia to water collected from Lake Loskop
– mRNA expression of a selection of genes associated with the thyroid system
Acknowledgements
References
•
Brion, F., Palazzi, X., Triffault, G., Laillet, B., Porcher, J. M., Garric, J., Tyler, C. R. and Flammarion, P. 2002. Reproductive effects of
exposure of various life stages of zebrafish. R&D TECHNICAL REPORT P2-T04/1 103
•
Casals-Casas, C., and Desvergne, B. 2011. Endocrine Disruptors: From endocrine to metabolic disruption. Annual Review of Physiology
73:135-162.
•
Jobling, S., Nolan, M., Tyler, C. R., Brighty, G., and Sumpter, J. P. 1998. Widespread sexual disruption in wild fish. Environmental Science
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•
Kid, K. A., Blanchfield, P. J., Mills, K. H., Palace, V. P., Evans, R. E., Lazorchak, J. M., and Flick, R. W. 2007. Collapse of a fish population
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•
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Pfaffl, M. W. 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research 29:e45.
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Woodling, J. D., Lopez, M. E., Maldonado, T. A., Norris, D. O., Vajda, A. M. 2006. Intersex and other reproductive disruption of fish in
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•
Yan, W., Zhou, Y., Yang, J., Li, S., Hu, D., Wang, J., Chen, J. , Li, G. 2012. Waterborne exposure to microcystin-LR alters thyroid hormone
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